ERK Activation Waves During MDCK Tissue Migration: Biosensor Analysis of Upstream and Parallel Effector Proteins

Abstract

During cellular migration, MDCK cells have been shown to exhibit waves of the protein ERK in the direction opposite the leading edge of movement. However, drivers of this wave as well as mechanisms for coupling ERK activation from one cell to the next have not yet been determined. ERK functions in parallel with PI3K and PLCγ and downstream of EGFR, which is driven by ADAM17. Live-cell fluorescent biosensors allow for the observation of transient, permissive, and oscillatory dynamics of these five key proteins in the ERK signaling cascade at the single-cell level. This research provides the first instance of using biosensors throughout the EGFR-ERK pathway during cellular migration to examine activity dynamics. These biosensors were qualitatively analyzed based on fluorophore localization within each cell, and quantitatively through the segmentation and analysis of fluorescent patterns in migrating tissues. In migration experiments, no dynamics were observed in any proteins upstream or in parallel with ERK through qualitative or quantitative analysis. Additionally, ERK exhibited a consistent density-dependent response during migration. Based on these experiments, EGFR is a permissive input into the ERK signaling cascade, leaving PI3K and PLCγ expression unaffected during migration. As a result, future experimentation should explore ERK regulatory proteins and related signaling pathways such as the Wnt pathway to determine inputs into ERK leading to waves.